This project aims to obtain an understanding of the nature, variability and evolution of the unusual genome of the spirochete bacteria (genus Borrelia) that cause human Lyme disease. Many of the genes that are thought to encode host-interaction genes in Borrelia are encoded on the numerous extrachromosomal DNA elements (plasmids) that these bacteria carry. Most of these "plasmids" (21 different ones in the only isolate to be exhaustively studied) may be present in nearly all natural isolates, and so could be thought of as "mini-chromosomes." Current evidence indicates that at least ten of the linear plasmids of the North American Lyme agent bacteria, Borrelia burgdorferi, are in the midst of a "rapid evolutionary spurt," as is evidenced by the presence in the one individual studied in detail, of many recent duplicative rearrangements and mutationally decaying duplicates of genes that are thought to be important to the organism. This application proposes to compare the complete nucleotide sequences of the plasmids several B. burgdorferi isolates, a B. garinii and a B. afzelii isolate (causative agents of Lyme disease in Eurasia), and isolates of a very closely related species that does not cause Lyme disease (B. bissettii). In addition, the chromosome of B. garinii will be sequenced. This "comparative genomics" approach is a powerful way to begin to deduce which plasmid genes are important in causing Lyme disease as indicated by their conservation among Lyme causative isolates, to deduce other general similarities and differences among the species under study, as well as to understand the mechanisms involved in the "rapid evolutionary changes" mentioned above. A longer term objective is to use this information to study a larger panel of independent strain isolates to understand the nature of the plasmid gene pool in the Lyme disease Borreliae, as well as to understand the relative rates of the "rapid evolution" (above), plasmid spread, and bacterial clonal expansion in the B. burgdorferi population. These findings will impact the study of the molecular pathogenesis of Lyme disease in many ways.